The successful elimination of pathogens, neoplastic cells, or self-reactive immune mechanisms following prophylactic or therapeutic immunization depends to a large extent on the ability of the host's immune system to become activated in response to the immunization and mount an effective response, preferably with minimal injury to healthy tissue.
The rational design of vaccines initially involves identification of immunological correlates of protection—the immune effector mechanism(s) responsible for protection against disease—and the subsequent selection of an antigen that is able to elicit the desired adaptive response. Once this appropriate antigen has been identified, it is essential to deliver it effectively to the host's immune system.
In the design of effective vaccines, immunological adjuvants serve as critical components, which accelerate, prolong, and/or enhance an antigen-specific immune response as well as provide the selective induction of the appropriate type of response.
New vaccines are presently under development and in testing for the control of various neoplastic, autoimmune and infectious diseases, including human immunodeficiency virus (HIV) and tuberculosis. In contrast to older vaccines which were typically based on live-attenuated or non-replicating inactivated pathogens, modem vaccines are composed of synthetic, recombinant, or highly purified subunit antigens. Subunit vaccines are designed to include only the antigens required for protective immunization and are believed to be safer than whole-inactivated or live-attenuated vaccines. However, the purity of the subunit antigens and the absence of the self-adjuvanting immunomodulatory components associated with attenuated or killed vaccines often result in weaker immunogenicity.
The immunogenicity of a relatively weak antigen can be enhanced by the simultaneous or more generally conjoined administration of the antigen with an “adjuvant”, usually a substance that is not immunogenic when administered alone, but will evoke, increase and/or prolong an immune response to an antigen. In the absence of adjuvant, reduced or no immune response may occur, or worse the host may become tolerized to the antigen.
Adjuvants can be found in a group of structurally heterogeneous compounds (Gupta et al., 1993, Vaccine, 11:293-306). Classically recognized examples of adjuvants include oil emulsions (e.g., Freund's adjuvant), saponins, aluminium or calcium salts (e.g., alum), non-ionic block polymer surfactants, lipopolysaccharides (LPS), mycobacteria, tetanus toxoid, and many others. Theoretically, each molecule or substance that is able to favor or amplify a particular situation in the cascade of immunological events, ultimately leading to a more pronounced immunological response can be defined as an adjuvant.
In principle, through the use of adjuvants in vaccine formulations, one can (1) direct and optimize immune responses that are appropriate or desirable for the vaccine; (2) enable mucosal delivery of vaccines, i.e., administration that results in contact of the vaccine with a mucosal surface such as buccal or gastric or lung epithelium and the associated lymphoid tissue; (3) promote cell-mediated immune responses; (4) enhance the immunogenicity of weaker immunogens, such as highly purified or recombinant antigens; (5) reduce the amount of antigen or the frequency of immunization required to provide protective immunity; and (6) improve the efficacy of vaccines in individuals with reduced or weakened immune responses, such as newborns, the aged, and immunocompromised vaccine recipients.
Although little is known about their mode of action, it is currently believed that adjuvants augment immune responses by one of the following mechanisms: (1) increasing the biological or immunologic half-life of antigens (see, e.g., Lascelles, 1989, Vet. Immunol. Immunopathol., 22: 15-27; Freund, 1956, Adv. Tuber. Res., 7: 130-147); (2) improving antigen delivery to antigen-presenting cells (APCs), as well as antigen processing and presentation by the APCs (see, e.g., Fazekas de St. Groth et al., Immunol. Today, 19: 448-454, 1998), e.g., by enabling antigen to cross endosomal membranes into the cytosol after ingestion of antigen-adjuvant complexes by APCs (Kovacsovics-Bankowski et al., Science, 1995, 267: 243-246); (3) mimicking microbial structures leading to improved recognition of microbially-derived antigens by the pathogen-recognition receptors (PRRs), which are localized on accessory cells from the innate immune system (Janeway, 1989, Cold Spring Harbor Symp. Quant. Biol., 54:1-13; Medzhitov, 1997, Cell, 91:295-298; Rook, 1993, Immunol. Today, 14:95-96); (4) mimicking danger-inducing signals from stressed or damaged cells which serve to initiate an immune response (see, e.g., Matzinger, 1994, Annu. Rev. Immunol., 12:991-209), (5) inducing the production of immunomodulatory cytokines (see, e.g., Nohria, 1994, Biotherapy, 7:261-269; Iwasaki et al., 1997, J. Immunol., 158:4591-4601; Maecker et al., 1997, Vaccine, 15:1687-1696); (6) biasing the immune response towards a specific subset of the immune system (e.g., generating Th1- or Th2-polarized response [see below], etc.) (Janssen et al., Blood, 97:2758-2763, 2001; Yamamoto et al., Scand. J. Immunol., 53:211-217, 2001; Weiner G. J., J. Leukoc. Biol., 68:455-63, 2000; Lucey, Infect. Dis. Clin. North Am., 13:1-9, 1999), and (7) blocking rapid dispersal of the antigen challenge (the “depot effect”) (Hood et al., Immunology, Second Ed., 1984, Benjamin/Cummings: Menlo Park, Calif.; St Clair et al., Proc. Natl. Acad. Sci. U.S.A., 96:9469-9474, 1999; Ahao et al., J. Pharm. Sci., 85:1261-1270, 1996; Morein et al., Vet. Immunol. Immunopathol., 54:373-384, 1996). (See also reviews by Schijns, Curr. Opin. Immunol., 12: 456-463, 2000; Vogel, Clin. Infect. Dis., 30 [Suppl. 3]: S266-70, 2000; Singh and O'Hagan, Nature Biotechnol., 17: 1075-81, 1999; Cox and Coulter, Vaccine, 15: 248-256, 1997).
Recent observations strongly suggest that endogenously produced cytokines act as essential communication signals elicited by traditional adjuvants. The redundancy of the cytokine network makes it difficult to ascribe the activity of a particular adjuvant to one or more cytokines. Cytokines crucial for immunogenicity may include the proinflammatory (Type 1) substances: interferon (IFN)-α/β, tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-6, IL-12, IL-15 and IL-18, which influence antigen presentation. Others may act more downstream during clonal expansion and differentiation of T and B cells, with IL-2, IL-4 and IFN-γ as prototypes (Brewer et al., 1996, Eur. J. Immunol., 26:2062-2066; Smith et al., 1998, Immunology, 93:556-562). Adjuvants that enhance immune responses through the induction of IFN-γ and delayed-type hypersensitivity also elicit the production of IgG subclasses that are the most active in complement-mediated lysis and in antibody-dependent cell-mediated-cytotoxicity effector mechanisms (e.g., IgG2a in mice and IgG1 in humans) (Allison, Dev. Biol. Stand., 1998, 92:3-11; Unkeless, Annu. Rev. Immunol., 1988, 6:251-81; Phillips et al., Vaccine, 1992, 10:151-8).
Clearly, some adjuvants may perform more than one function. For example, purified microbial components such as LPS or extracts of Toxoplasma gondii rapidly increase not only the number of antigen-presenting dendritic cells (DC) and their migration but also IL-12 production (Souza et al., 1997, J. Exp. Med., 186:1819-1829).
As different adjuvants may have diverse mechanisms of action, their being chosen for use with a particular vaccine may be based on the route of administration to be employed, the type of immune responses desired (e.g., antibody-mediated, cell-mediated, mucosal, etc.), and the particular inadequacy of the primary antigen.
The benefit of incorporating adjuvants into vaccine formulations to enhance immunogenicity must be weighed against the risk that these agents will induce adverse local and/or systemic reactions. Local adverse reactions include local inflammation at the injection site and, rarely, the induction of granuloma or sterile abscess formation. Systemic reactions to adjuvants observed in laboratory animals include malaise, fever, adjuvant arthritis, and anterior uveitis (Allison et al., Mol. Immunol., 1991, 28:279-84; Waters et al., Infect. Immun., 1986, 51:816-25). Such reactions often are caused by the interaction of the adjuvant and the antigen itself, or may be due to the type of response to a particular antigen the adjuvant produces, or the cytokine profile the adjuvant induces.
Thus, many potent immunoadjuvants, such as Freund's Complete or Freund's Incomplete Adjuvant, are toxic and are therefore useful only for animal research purposes, not human vaccinations. Currently, aluminum salts and MF59 are the only vaccine adjuvants approved for human use. Of the novel adjuvants under evaluation, immunostimulatory molecules such as the lipopolysaccharide-derived MPL and the saponin derivative QS-21 appear most promising, although doubts have been raised as to their safety for human use. Preclinical work with particulate adjuvants, such as the MF59 microemulsion and lipid-particle immuno-stimulating complexes (ISCOMs), suggest that these molecules are also themselves potent elicitors of humoral and cellular immune responses. In addition, preclinical data on CpG oligonucleotides appear to be encouraging, particularly with respect to their ability to manipulate immune responses selectively. While all these adjuvants show promise, the development of more potent novel adjuvants may allow novel vaccines to be developed and both novel and existing vaccines to be used as therapeutic as well as improved prophylactic agents.
Recently, a novel lymphoid lineage, natural killer T (NKT) cells, distinct from mainstream T cells, B cells and NK cells, has been identified (Arase et al., 1992, Proc. Natl Acad. Sci. USA, 89:6506; Bendelac et al., 1997, Annu. Rev. Immunol., 15:535). These cells are characterized by co-expression of NK cell receptors and semi-invariant T cell receptors (TCR) encoded by Vα14 and Jα281 gene segments in mice and Vα24 and JαQ gene segments in humans. The activation of NKT cells in vivo promptly induces a series of cellular activation events leading to the activation of innate cells such as natural killer (NK) cells and dendritic cells (DC), the activation of adaptive cells such as B cells and T cells, the induction of co-stimulatory molecules and the abrupt release of cytokines such as interleukin-4 (IL-4) and interferon-γ (IFN-γ) (Burdin et al., Eur. J. Immunol. 29: 2014-2025, 1999; Carnaud et al., J. Immunol., 163: 4647-4650, 1999; Kitamura et al., J. Exp. Med., 189: 1121-1128, 1999; Kitamura et al., Cell Immunol., 199: 37-42, 2000; Aderem and Ulevitch, Nature, 406: 782-787, 2000). In addition, activated NKT cells can themselves bring about killing mediated by Fas and perforin. The full activation cascade can be recruited by the engagement of NKT TCR. Alternatively, powerful T-helper-cell type 1 (Th1) functions can be selectively triggered by cytokines such as interleukin-12 (IL-12) released by infected macrophages or DC. These functions are believed likely to be correlated with the important role of NKT cells in conditions such as autoimmune diabetes, rejection of established tumours or the prevention of chemically induced tumours (Yoshimoto et al, 1995, Science, 270: 1845; Hammond et al., J. Exp. Med., 187: 1047-1056, 1998; Kawano et al., 1998, Proc. Natl. Acad. Sci. USA, 95: 5690; Lehuen et al., J. Exp. Med., 188: 1831-1839, 1998; Wilson et al., Nature, 391: 177-181, 1998; Smyth et al., J. Exp. Med., 191: 661-668, 2000). Finally, NKT cells are thought to contribute to antimicrobial immunity through their capacity to influence the Th1-Th2 polarization (Cui et al., J. Exp. Med., 190: 783-792, 1999; Singh et al., J. Immunol., 163: 2373-2377, 1999; Shinkai and Locksley, J. Exp. Med., 191: 907-914, 2000). These cells are therefore implicated as key effector cells in innate immune responses. However, the potential role of NKT cells in the development of adaptive immune responses remains unclear.
Recently, it was demonstrated that NKT cells can be activated both in vitro and in vivo by α-galactosyl-ceramide (α-GalCer), a glycolipid originally extracted from Okinawan marine sponges (Natori et al., Tetrahedron, 50: 2771-2784, 1994), or its synthetic analog KRN 7000 [(2S,3S,4R)-1—O-(α-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4,-octadecanetriol] which can be obtained from Pharmaceutical Research Laboratories, Kirin Brewery (Gumna, Japan) or synthesized as described previously (see, e.g., Kobayashi et al., 1995, Oncol. Res., 7:529-534; Kawano et al., 1997, Science, 278:1626-9; Burdin et al., 1998, J. Immunol., 161:3271; Kitamura et al., 1999, J. Exp. Med., 189:1121; U.S. Pat. No. 5,936,076). Thus, it was shown that α-GalCer can stimulate NK activity and cytokine production by NKT cells and exhibits potent antitumor activity in vivo (Kawano et al., 1997, supra; Kawano et al., 1998, Proc. Natl Acad. Sci. USA, 95:5690; Kitamura et al., 1999, supra). Kitamura et al. (1999, supra) demonstrated that the immunostimulating effect of α-GalCer was initiated by CD40-CD40L-mediated NKT-DC interactions. As the immunoregulatory functions of α-GalCer were absent in both CD1d−/− and NKT-deficient mice, this indicates that α-GalCer has to be presented by the MHC class I-like molecule CD1d.
CD1 is a conserved family of non-polymorphic genes related to MHC that seems to have evolved to present lipid and glycolipid antigens to T cells and in this way participates in both an innate and an adaptive pathway of antigen recognition (reviewed by Park and Bendelac, Nature, 406: 788-792, 2000; see also Calabi et al., Eur. J. Immunol., 19: 285-292, 1989; Porcelli and Modlin, Annu. Rev. Immunol., 17: 297-329, 1999). It comprises up to five distinct genes (isotypes) that can be separated into two groups on the basis of sequence homology. Group 1, which comprises CD1a, CD1b, CD1c and CD1e, is present in humans but absent from mouse and rat. Group2, which includes CD1d, is found in all species studied so far, including humans.
CD1 isotypes are expressed selectively by antigen-presenting cells such as dendritic cells (DCs), macrophages and subsets of B cells, but apart from CD1d expression in hepatocytes they are generally not expressed in solid tissues (Porcelli et al., supra; Bendelac et al., Annu. Rev. Immunol., 15: 535-562, 1997).
α-GalCer is recognized in picomolar concentrations by those among mouse and human CD1d-restricted lymphocytes that express a semi-invariant TCR and exert potent effector and regulatory functions (Kawano et al., Science, 278: 1626-1629, 1997). CD1d/α-GalCer complex is, in turn, recognized by the antigen receptors of mouse Vα14 and human Vα24 natural killer T (NKT) cells (Bendelac et al., Science, 268: 863-865, 1995; Bendelac et al., Annu. Rev. Immunol., 15: 535-562, 1997; Park et al., Eur. J. Immunol., 30: 620-625, 2000).
Upon binding to CD1d, α-GalCer was demonstrated to activate murine NKT cells both in vivo and in vitro (Kawano et al., 1997, Science, 278:1626-1629; Burdin et al., 1998, J. Immunol., 161:3271-3281), and human NKT cells in vitro (Spada et al., 1998, J. Exp. Med., 188:1529-1534; Brossay et al., 1998, J. Exp. Med. 188:1521-1528). For example, α-GalCer was shown to display NKT-mediated anti-tumor activity in vitro by activating human NKT cells (Kawano et al., 1999, Cancer Res., 59:5102-5105).
In addition to α-GalCer, other glycosylceramides having α-anomeric conformation of sugar moiety and 3,4-hydroxyl groups of the phytosphingosine (such as α-glucosylceramide [α-GlcCer], Galα1-6Galα1-1′Cer, Galα1-6Glcα1-1′Cer, Galα1-2Galα1-1′Cer, and Galβ1-3Galα1-1′Cer) have been demonstrated to stimulate proliferation of Vα14 NKT cells in mice, although with lower efficiency (Kawano et al., Science, 278: 1626-1629, 1997). By testing a panel of α-GalCer analogs for reactivity with mouse Vα14 NKT cell hybridomas, Brossay et al. (J. Immunol., 161: 5124-5128, 1998) determined that nearly complete truncation of the α-GalCer acyl chain from 24 to 2 carbons does not significantly affect the mouse NKT cell response to glycolipid presented by either mouse CD1 or its human homolog.
It has been also demonstrated that in vivo administration α-GalCer not only causes the activation of NKT cells to induce a strong NK activity and cytokine production (e.g., IL-4, IL-12 and IFN-γ) by CD1d-restricted mechanisms, but also induces the activation of immunoregulatory cells involved in acquired immunity (Nishimura et al., 2000, Int. Immunol., 12: 987-994). Specifically, in addition to the activation of macrophages and NKT cells, it was shown that in vivo administration of α-GalCer resulted in the induction of the early activation marker CD69 on CD4+ T cells, CD8+ T cells, and B cells (Burdin et al., 1999, Eur. J. Immunol. 29: 2014; Singh et al., 1999, J. Immunol. 163: 2373; Kitamura et al., 2000, Cell. Immunol. 199:37; Schofield et al., 1999, Science 283: 225; Eberl et al., 2000, J. Immunol., 165:4305-4311). These studies open the possibility that α-GalCer may play an equally important role in bridging not only innate immunity mediated by NKT cells, but also adaptive immunity mediated by B cells, T helper (Th) cells and T cytotoxic (Tc) cells.
Due to the identification of new tumor-specific antigens and realization that the immune system plays a critical role in the prevention of cancer and the control of tumor growth, in recent years, there has been a renewed interest in the development of therapeutic cancer vaccines (e.g., to reduce tumor burden and control metastasis).
The demonstration that in vivo engagement of NKT cells by their glycolipid ligand α-GalCer rapidly induces a cascade of cellular activation that involves elements common to innate and adaptive immunity as well as the generation of tumor-specific cytotoxic T cells (Nishimura et al., 2000, supra) suggests that α-GalCer administration may generally affect not only the speed and strength but also the type of subsequent immune responses, in particular, those directed against tumor cells. Indeed, Kabayashi et al. (1995, Oncol. Res., 7: 529-534) discovered that a synthetic form of α-GalCer (KRN 7000) had stronger antimetastatic activities in B 16-bearing mice than biological response modifiers such as OK432 and Lentinan and a chemotherapeutic agent Mitomycin C. In these experiments, 60% of mice bearing tumors were cured by treatment with 100 μg/kg KRN7000. KRN7000 was also shown to induce a pronounced tumor-specific immunity in mice with liver metastasis of murine T-lymphoma EL-4 cells (Nakagawa et al., Oncol. Res., 10: 561-568, 1998) or Colon26 cells (Nakagawa et al., Cancer Res., 58: 1202-1207, 1998). Furthermore, the administration of α-GalCer to mice was found to inhibit the development of hepatic metastasis of primary melanomas (Kawano et al., 1998, Proc. Natl. Acad. Sci. USA, 95: 5690-5693).
The data presented above have led the present inventors to a hypothesis that the glycosylceramide-induced NKT cell responses may also contribute to immune responses involved in combating various infections. Indeed, the present inventors and co-workers have recently observed that the administration of α-GalCer to mice resulted rapidly in strong anti-malaria activity, inhibiting the development of intra-hepatocytic stages of the rodent malaria parasites, P. yoeli and P. berghei (Gonzalez-Aseguinolaza et al., 2000, Proc. Natl. Acad. Sci. USA, 97: 8461-8466). The administration of α-GalCer alone to mice lacking either CD1d or Vα14 NKT cells, however, failed to protect them against malaria, indicating that the anti-malaria activity of α-GalCer requires both NKT cells and the expression of CD1d. Furthermore, α-GalCer was unable to inhibit parasite development in the liver of mice lacking either IFN-γ or the IFN-γ receptor, indicating that the anti-malaria activity of the glycolipid is primarily mediated by IFN-γ.
In light of the data on the NKT-mediated anti-tumor and anti-parasite activity of α-GalCer, it has been proposed that this glycolipid is a potent inducer of protective immune responses (see, e.g., Park and Bendelac, supra). The present inventors have significantly expanded these hypotheses by conceiving and demonstrating for the first time that α-GalCer and related glycosylceramides can be employed not just as antigens but also as adjuvants capable of enhancing and/or extending the duration of the protective immune responses induced by other antigens. This is an unexpected discovery, because α-GalCer-mediated NKT cell activation results in the complete elimination of malaria-infected cells, thus eliminating the source of antigen necessary for the development of an adaptive immune response. In fact, the administration of α-GalCer two days before immunization with irradiated sporozoites almost completely abolishes sporozoites-induced protection. Therefore, in order to use α-GalCer as an adjuvant, the timing of the administration in relation to the antigen given is very important.
Accordingly, the present invention provides for the first time methods and compositions for enhancing and/or extending the duration of the immune response against an antigen in a mammal, notably a human, involving the conjoint immunization of the mammal with (i) an antigen and (ii) an adjuvant comprising glycosylceramide, in particular, α-GalCer.
Importantly, in addition to its ability to stimulate immune responses, it has been demonstrated that α-GalCer, independently of its dosage, does not induce toxicity in rodents and monkeys (Nakagawa et al., 1998, Cancer Res., 58: 1202-1207). Moreover, although a recent study showed the transient elevation of liver enzyme activities immediately after α-GalCer treatment in mice, suggesting a minor liver injury (Osman et al., 2000, Eur. J. Immunol., 39: 1919-1928), human trials are currently being conducted using α-GalCer to treat cancer patients without a notable complication (Giaccone et al., 2000, Abstract. Proc. Amer. Soc. Clin. Oncol., 19: 477a). Finally, unlike many other newly developed adjuvants (see below), α-GalCer and related glycosylceramides can be produced synthetically with reasonable yields and efficiency (see, e.g., U.S. Pat. No. 5,936,076). All of these factors make glycosylceramides and, in particular α-GalCer, desirable adjuvant candidates.
In contrast to α-GalCer and related glycosylceramides, conventional vaccine delivery systems and the adjuvants approved for human use, aluminium salts and MF59 (Singh and O'Hagan, Nat. Biotechnol., 17: 1075-1081, 1999), are poor at inducing CD8+ T cell responses. Although certain novel adjuvants, such as purified saponins, immunostimulatory complexes, liposomes, CpG DNA motifs, and recombinant attenuated viruses (e.g., adenovirus, Sindbis virus, influenza virus, and vaccinia virus), have been shown to improve the antigen specific cellular immune responses over those induced by the same antigen given alone or in combination with standard alum adjuvants (Newman et al., J. Immunol., 1992; 148:2357-2362; Takahashi et al., Nature, 1990, 344:873-875; Babu et al., Vaccine, 1995, 13:1669-1676; Powers et al., J. Infect. Dis., 1995, 172:1103-7; White et al., Vaccine, 1995, 13:1111-1122; Krieg et al., Trends Microbiol., 6: 23-27, 1998; Rodrigues et al., J. Immunol., 158: 1268-1274, 1997; Tsuji et al., J. Virol., 72: 6907-6910, 1998; Li et al., Proc. Natl. Acad. Sci. USA, 90: 5214-52188, 1993), none of the currently available adjuvants combine low toxicity in humans, cost-efficiency of production and the ability to efficiently stimulate the immune system.
The development of an adaptive immune response is a multifactorial phenomenon, in which many elements participate. In this regard, α-GalCer-activated NKT cells induce the activation of many of the elements involved in the development of the adaptive immune response, such as antigen presenting cells (APC), B cells, T helper (Th) cells and T cytotoxic (Tc) cells. Therefore, theoretically, α-GalCer could be an ideal immunomodulator. Additional advantage is that α-GalCer can be administered and activate the immune system via many different routes, including oral, subcutaneous, and intramuscular routes, which are suitable for human use. Finally, it has been shown that α-GalCer does not induce toxicity in rodents and monkeys (Nakagawa et al., Cancer Res., 58:1202-1207, 1998).
Accordingly, there is a great need in the art to develop new adjuvants that would combine low toxicity and easy availability with the ability to enhance and/or prolong the antigen-specific immune responses to a significant degree. The present invention addresses these and other needs in the art by providing glycosylceramides, a novel group of adjuvants with superior properties. Such adjuvants can improve prophylactic and/or therapeutic vaccines for the treatment of various infections and cancers.